This project studies the physiology and development of calcium regulation during neuronal activity, with special emphasis on the dendrites and dendritic spines of central nervous system neurons. Previously, Ca imaging and analytical studies on dendrites of CA3 neurons in hippocampal slice cultures had characterized a subset of endoplasmic reticulum (ER) as the major calcium-sequestering organelle in the time frame of seconds to 15 minutes following afferent synaptic activity. In contrast, mitochondrial calcium uptake operates in parallel with ER sequestration only over the first minute following synaptic activity. A new study reveals a different mode of Ca sequestration in sympathetic neurons. In these neurons, measurements of total Ca (x-ray microanalysis) in parallel with recordings of free cytosolic Ca2+ transients (fura- 2), following depolarizations that evoke defined Ca2+ transients, provide direct evidence for sustained (up to 5 min) and robust calcium sequestration within mitochondria, while ER Ca was little changed. This mitochondrial Ca accumulation is large, time dependent (more than 10 mmol/kg total Ca concentration after a 45 sec depolarization), reversible and sensitive to agents that disrupt mitochondrial transport pathways. Previously we had shown that estradiol doubles dendritic spine density of hippocampal neurons via its effect on inhibitory hippocampal interneurons. These results provided an attractive explanation for the observation that estrogen affects cognition and plasticity in adult organisms. New studies with hippocampal neurons in culture reveal that brain-derived neurotrophic factor (BDNF), which has been implicated in the regulation of GABAergic neurons, is downregulated by 40% following estradiol treatment. This, in turn, increases excitatory tone in these neurons, leading to a twofold increase in spines. These observations reveal a functional link between estradiol and BDNF (as a regulator of GABAergic interneurons) in the activity-dependent enhancement of dendritic spines.